Division of Organic Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, Amsterdam, Noord-Holland, The Netherlands.
Methods Mol Biol. 2021;2323:171-197. doi: 10.1007/978-1-0716-1499-0_13.
The method development to analyze direct RNA-protein interaction is of high importance as not many homogeneous assay formats are available.The discovery of fluorescent light-up aptamers (FLAPs), short RNA aptamers that switch the fluorescence of small, cell-permeable, and nontoxic organic chromophores on, paved the road for their utilization in direct RNA -protein interactions. The combination with fluorescent proteins as biological fluorophores enabled the development of homogeneous assays that are in principle even encodable on genomic level.Here the rules and methods to design a homogeneous in vitro assay for the detection and quantification of a direct RNA -protein interaction will be described. The design and application of a homogeneous assay to observe and quantify the interaction of the Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) with its binding RNA sequence (pp7-RNA) will be shown. For this, the Spinach-DFHBI aptamer as RNA fusion and the red fluorescent mCherry as protein fusion was used.The methods presented here do not require any chemical modification of proteins or RNAs which make them relatively easy to use and to adopt on other systems. As all fluorophores are fusion tags to the according biomolecules, standard cloning strategies and molecular biology technologies are sufficient and make this method available for a broad community of researchers.
开发用于分析直接 RNA-蛋白质相互作用的方法非常重要，因为可用的同质测定方法并不多。荧光亮发适体 (FLAPs) 的发现，即短 RNA 适体，可使小分子、细胞通透性和非毒性的有机发色团的荧光发生转变，为其在直接 RNA-蛋白质相互作用中的应用铺平了道路。与荧光蛋白结合作为生物荧光团，使得开发同质测定方法成为可能，这些方法在原理上甚至可以在基因组水平上进行编码。这里将描述设计用于检测和定量直接 RNA-蛋白质相互作用的同质体外测定的规则和方法。将展示一种用于观察和定量铜绿假单胞菌噬菌体衣壳蛋白 7 (PP7)与其结合 RNA 序列 (pp7-RNA) 相互作用的同质测定的设计和应用。为此，使用了 Spinach-DFHBI 适体作为 RNA 融合物和红色荧光 mCherry 作为蛋白质融合物。这里介绍的方法不需要对蛋白质或 RNA 进行任何化学修饰，因此相对容易使用和在其他系统中采用。由于所有荧光团都是相应生物分子的融合标签，因此标准的克隆策略和分子生物学技术就足够了，这使得这种方法可以被广泛的研究人员使用。
